Ligand-directed selective protein modification based on local single-electron-transfer catalysis

Article abstract of DOI:10.1002/anie.201303831

A photocatalyst ([Ru(bpy)₃]²⁺) bound to a protein ligand was essential for the title method. Local single-electron transfer from the catalyst resulted in the formation of tyrosyl radicals. N′-Acetyl-N,N- dimethyl-1,4-phenylenediamine was used as the tyrosyl radical trapping agent and used in a radical addition to afford selective modification of the target protein. Copyright

Full text of DOI:10.1002/anie.201303831

Angewandte
Chemie
Communications
Protein Modification
S. Sato, H. Nakamura*
&&&& — &&&&
Ligand-Directed Selective Protein
Modification Based on Local Single-
Electron Transfer Catalysis
A photocatalyst ([Ru(bpy)3]2+) bound to
a protein ligand was essential for the title
method. Local single-electron transfer
from the catalyst resulted in the forma-
tion of tyrosyl radicals. N’-acetyl-N,N-
dimethyl-1,4-phenylenediamine was used
as the tyrosyl radical trapping agent and
reacted in a radical addition resulting in
the selective modification of the target
protein.
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DOI: 10.1002/anie.201303831
Protein Modification
Ligand-Directed Selective Protein Modification Based on Local Single-
Electron Transfer Catalysis**
Shinichi Sato and Hiroyuki Nakamura*
Techniques for the visualization of target proteins in living
systems are highly important to investigate the function,
dynamics, localization, and crosstalk of individual proteins.[1]
For this purpose, GFP fusion tags and monoclonal anti-
bodies[2] are widely used for conventional protein labeling in
molecular biology. Although these are useful methods for the
real-time monitoring of target proteins in living systems,
genetic manipulation is often necessary, and the conjugation
of these proteins with relatively large labeling groups some-
times results in improper biophysical functions owing to
undesirable interactions with other molecules.
with ligand molecules specifically reacted with the nucleo-
philic amino acid side chains on the surfaces of target proteins
through an SN2-type reaction with the concomitant release of
the ligand molecules. Furthermore, they were the first to
demonstrate the catalytic target-selective modification using
ligand-tethered N,N-dimethylaminopyridine (DMAP) cata-
lysts (Figure 1A), which allowed highly efficient transfer of
The chemical modification of proteins with small-mole-
cule probes has received much interest as an alternative
powerful method for the study of individual proteins in their
native environments. The key to this chemical modification is
a bioorthogonal chemical reaction that enables the rapid and
selective bioconjugation of proteins with nonnatural func-
tional groups under physiological conditions. The most widely
utilized bioorthogonal chemical reactions rely on electro-
philic reagents that target nucleophilic amino acids, such as
lysine and cysteine. In addition, the lysine-specific reductive
alkylation using an iridium catalyst,[3] the conversion of
cysteine into dehydroalanine,[4] and the radical addition
reaction of cysteine with an alkene[5] were recently developed.
Furthermore, remarkable attention has been paid to the
modification of aromatic amino acids, such as tyrosine and
tryptophan, by transition-metal-mediated processes,[6–8] the
three-component Mannich reaction,[9] the click-like ene-type
reaction,[10] the oxidative modification using cerium(IV)
ammonium nitrate,[11] and the use of formylbenzene diazo-
nium reagents.[12]
Although various bioorthogonal chemical reactions have
been reported, there are still few reports regarding selective
chemical modifications of native target proteins. Popp and
Ball recently reported the site-specific protein modification of
aromatic side chains with dirhodium metallopeptide cata-
lysts.[13,14] Hamachi and co-workers pioneered a ligand-
directed protein labeling method in which an electrophilic
phenylsulfonate ester[15] or imidazole[16] group conjugated
Figure 1. Ligand-directed selective protein modification. A) Acyl-trans-
fer DMAP catalyst promotes the acylation of the nucleophilic regions
of the target protein through an SN2-type reaction. B) Local SET
catalyst (LSC; this work) promotes the generation of tyrosyl radicals
that react with tyrosyl radical trapping agents (TRTs) in the local
environment of the probe on the target protein in the presence of
visible light.
acyl donor probes to the nucleophilic regions of the targeted
protein through an SN2-type reaction.[17,18] Herein, we report
the development of a ligand-directed selective protein
modification method based on local single-electron transfer
catalysis (Figure 1B). In this method, a single-electron trans-
fer (SET) with a photocatalyst,[19,20] such as the ruthenium
tris(2,2’-bipyridyl) complex ([Ru(bpy)3]2+), is essential for the
generation of tyrosyl radicals[21,22] that react with tyrosyl
radical trapping agents (TRTs) containing an N’-acyl-N,N-
dimethyl-1,4-phenylenediamine through a catalytic oxidative
radical addition reaction.
[*] Dr. S. Sato, Prof. H. Nakamura
Department of Chemistry, Faculty of Science, Gakushuin University
Mejiro, Tokyo 171-8588 (Japan)
E-mail: hiroyuki.nakamura@gakushuin.ac.jp
[**] This work was partially supported by a Grant-in-Aid for Scientific
Research on Innovative Areas “Chemical Biology of Natural
Products” from The Ministry of Education, Culture, Sports, Science
and Technology (Japan).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201303831.
2
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We first examined TRTs suitable for the SET-based
addition reaction using angiotensin II as the model peptide.
Among the compounds examined, N’-acetyl-N,N-dimethyl-
1,4-phenylenediamine (1; Table 1) was found to be the most
suitable TRT for this reaction, and the monoadduct to
angiotensin II was obtained in 50% yield in the presence of
the [Ru(bpy)3]Cl2 complex in Tris buffer (10 mm, pH 4.2) and
tions proceeded through the photoinduced oxidative SET
radical mechanism proposed in Scheme S1 in the Supporting
Information. MS/MS analysis of the mono- and bisadducts of
1 to angiotensin II indicated that all modifications were
implemented on tyrosine residues (Figures S1 and S2 in the
Supporting Information). To understand the binding mode
between 1 and tyrosine residues, the addition reaction of
1 with ethyl-N-acetyl-tyrosine amide, a model substrate, was
investigated. Structural analysis revealed that a carbon–
carbon bond was formed between the ortho-carbon atom of
the phenolic oxygen of ethyl-N-acetyl-tyrosine amide and the
ortho-carbon atom of the dimethylamino group of 1 (Fig-
ures S3 and S4 in the Supporting Information). We also
examined the reactivity of 1 to the tryptophan residue by
using the model peptide melittin. The observed oxidation of
the tryptophan residue with oxygen proceeded prior to the
addition with 1.[24–26] These results suggest that this method
can be used for the specific modification of tyrosine residues
in the target protein.
With the suitable conditions established, we applied this
tyrosine-residue-specific addition reaction to the modification
of a purified protein. We designed and synthesized fluores-
cent TRT 2 (Scheme S3 in the Supporting Information).
Bovine serum albumin (BSA), the model protein, was treated
with 2 under several conditions (Figure 2). Whereas BSA was
not modified in the presence of [Ru(bpy)3]Cl2 without 2 or
APS (lanes 1 and 2), successful modification was observed in
the presence of [Ru(bpy)3]Cl2 (10 mm), 2 (500 mm), and APS
(1 mm) under irradiation with visible light for 5 min (lane 3).
A slight modification of BSA with 2 was observed without
irradiation and [Ru(bpy)3]Cl2 (lanes 4 and 5), thereby
revealing that APS caused a photocatalyst-independent
modification reaction, which was considered to be one of
the undesired background reactions. This modification pro-
ceeded without APS, although an excess amount of [Ru-
Table 1: Optimization of the addition reaction of angiotensin II with
N’-acetyl-N,N-dimethyl-1,4-phenylenediamine (1).[a]
Entry [Ru(bpy)3]Cl2 Buffer Irradiation
Additive
–
1 mm APS 70 (9:1)
1 mm APS 95 (3:2)
Yield [%]
(mono-/
bisadduct)
pH
time [min]
1
2
3
4
1 mm
10 mm
1 mm
1 mm
6.0
7.4
7.4
7.4
15
5
1
55 (1:0)
1
1 mm APS
0
10 mm DTT
1 mm APS 10 (1:0)
1 mm APS 10 (1:0)
[b]
5
6
7
1 mm
–
1 mm
7.4
7.4
7.4
–
–
–
[b]
[b]
–
0
[a] Reaction conditions: angiotensin II (100 mm) and 1 (500 mm) in MES
(10 mm) buffer. All reactions were quenched with DTT (10 mm) and
analyzed by using MALDI-TOF MS. Each reaction was repeated several
times and the average ratios of the mono- and bisadducts are indicated.
[b] Incubated for 5 min without irradiation.
under irradiation with light (Table S1 in the Supporting
Information). Then, we tested different buffers and additives
for the addition reaction with TRT 1 under mild conditions
(pH 6.0–7.4) to prevent proteins from denaturation (Table 1
and Table S2 in the Supporting Information). The addition
reaction proceeded preferentially in 2-morpholinoethanesul-
fonic acid (10 mm, MES) buffer under irradiation with visible
light for 15 min (Table 1, entry 1). It was reported that SETof
the excited state *RuII to RuIII was accelerated in the presence
of oxidants, such as ammonium persulfate (APS).[21] There-
fore, we examined the effects of APS on the addition reaction
of angiotensin II (100 mm) with 1 (500 mm). The reaction
proceeded smoothly at pH 7.4 in the presence of APS (1 mm)
and a catalytic amount of [Ru(bpy)3]Cl2 (10 mm) under
irradiation with visible light for 5 min, giving the monoadduct
and the bisadduct (9:1 ratio) in 70% yield (Table 1, entry 2).
The use of 1 mm of [Ru(bpy)3]Cl2 accelerated the reaction
rate and both mono- and bisadducts were obtained in 95%
yield in a 3:2 ratio even under irradiation with visible light for
1 min (Table 1, entry 3). The reaction was abolished by
Figure 2. Modification of BSA with fluorescent TRT 2 under various
conditions. Fluorescence images and coomassie brilliant blue (CBB)-
stained images of SDS-PAGE gels, with the conditions for each lane
given above. [a] The exposure time for fluorescence detection in lanes
6–10 was longer (3.0 s) than that in lanes 1–5 (0.3 s; also see
Figure S7 in the Supporting Information).
addition of dithiothreitol (DTT),
a radical scavenger
(Table 1, entry 4). Both irradiation with visible light and the
[Ru(bpy)3]Cl2 complex were essential for the addition reac-
tion (Table 1, entries 5–7),[23] thus suggesting that the reac-
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Figure 4. Selective modification of CA in mouse erythrocyte lysates.
A) Structure of biotin-conjugated TRT 5. B) The reaction for the
selective modification of CA was performed in MES (10 mm, pH 6.0)
using erythrocyte lysates from 6-week-old female mice. The left image
shows visualization of biotinylated proteins with a streptavidin–HRP
conjugate. The right image shows CBB staining of proteins in mouse
erythrocyte lysates.
Figure 3. Selective modification of carbonic anhydrase (CA) promoted
by the CA-ligand-conjugated ruthenium complex 3 as LSC in a mixture
of BSA and bovine CA. A) Structures of 3 and CA-binding competitor
4. B) Protein modification reactions were performed in a mixture of
BSA (5 mm) and CA (5 mm) in MES buffer (10 mm; pH 6.0) without
APS. Fluorescence images of modified proteins (upper) and whole
proteins with CBB stain (lower) are shown.
more than 100 times higher efficiency than BSA modification
(lane 3; 1 mm [Ru(bpy)3]Cl2 versus lane 4; 10 mm 3).[28] The
selective modification of CA was inhibited by the CA-binding
competitor 4, which was present in an amount that was 100
times that of LSC 3 (lane 5), thereby revealing that this
reaction was accelerated by the target protein–ligand inter-
action between CA and 3.[29] No protein modification of both
BSA and CA was observed in the absence of irradiation with
visible light or 2 (lanes 6 and 7).
Finally, we applied this target-selective protein modifica-
tion to native CA proteins in cell lysate using 3. CA
modification was performed in mouse erythrocytes that
originally expressed CAs. However, the lysates of erythro-
cytes contained a large amount of hemoglobin (14 kDa) and
the fluorescence of hemoglobin interfered with the fluores-
cence detection of CA modified with 2. Therefore, we
developed biotin-conjugated N,N-dimethyl-1,4-phenylenedi-
amine 5 as a TRT (Figure 4A) to detect the target protein by
using a streptavidin–horseradish peroxidase(HRP) conjugate.
No biotinylated proteins were detected with streptavidin–
HRP in the absence of 3, biotin-conjugated TRT 5, or
irradiation (Figure 4B, lanes 1–4). Those proteins, however,
were observed at 29 kDa, which corresponded to CA from the
cell lysate, in the presence of 3 and 5 under irradiation with
light for 15 min (lane 5), and this modification was inhibited
by the addition of 4 (lane 6).[30] These results clearly indicated
that SET-based protein modification using 3 and 5 specifically
occurred on the target protein CA despite the coexistence of
various kinds of proteins in the cell lysate. We also performed
(bpy)3]Cl2 (1 mm) was needed. [Ru(bpy)3]Cl2 and irradiation
with light were indispensable in this case (lanes 6–10). To
evaluate the biocompatibility of this reaction, double modi-
fication experiments of BSA and streptavidin were carried
out using PEG-conjugated TRT and tetramethylrhodamine-
conjugated maleimide or N-hydroxysuccinimide (NHS). The
electrophilic modifications proceeded even after the PEG
modification, thus suggesting that this SET-based tyrosine-
targeting method is compatible with the electrophilic meth-
ods targeting cysteine and lysine residues (see Figure S8 in the
Supporting Information).
To clarify whether this addition reaction based on local
SET is applicable to selective protein modification, we
designed and synthesized a ligand-conjugated ruthenium
complex as an LSC. Carbonic anhydrase (CA) and benzene-
sulfonamide were chosen as the model of the target protein
and its ligand, respectively.[27] When the SET-based radical
modification reaction was carried out in the presence of
[Ru(bpy)3]Cl2 using a mixture of BSA and CA, the extent of
modification of BSA was higher than of CA, because it had
a larger number of accessible tyrosine residues (Figure 3,
lanes 2 and 3). Meanwhile, benzenesulfonamide-conjugated
ruthenium complex 3, which was designed as a CA-targeting
LSC, promoted the selective modification of CA (lane 4) with
4
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[10] H. Ban, J. Gavrilyuk, C. F. Barbas, J. Am. Chem. Soc. 2010, 132,
this modification reaction in intact erythrocytes in MES-
buffered saline (10 mm MES (pH 6.0), 150 mm NaCl) and
again obtained similar results to those from the cell lysate
(Figure S11 in the Supporting Information), thus suggesting
that 5 and 3 could permeate the erythrocyte cell membrane.[31]
Therefore, this target-selective protein modification based on
local SET catalysis is applicable to the modification of native
proteins in an intracellular environment.
In conclusion, we have developed a target-selective
protein modification strategy based on local SET catalysis.
In cell lysates, tyrosyl radicals were selectively generated on
the target protein by SET from the photocatalyst in the local
environment of the ligand, and the radicals were trapped by
TRTs through the specific radical addition to tyrosine
residues. Although target-selective protein modifications in
cell lysates were achieved before with electrophilic reagents
using ligand-tethered DMAP catalysts, those methods were
limited to modifying the nucleophilic residues of the targeted
proteins, such as lysine and cysteine. The current method is
not only an alternative to the strategy using electrophilic
agents but can also be utilized for the identification of
proteins interacting with the target protein through a tyro-
sine–tyrosine SET addition reaction between the target
protein and its interaction partners induced by the LSC-
based generation of tyrosyl radicals. Studies with respect to
further applications are in progress.
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[23] The modifications induced by APS were observed in 10%
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Received: May 4, 2013
Published online: && &&, &&&&
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[28] The use of lower amounts of compound 3 resulted in lower
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CA.
[29] We examined the CA inhibitory activity of compounds 3, 4 and
[Ru(bpy)3]Cl2 according to the literature protocol reported by
J. A. Verpoorte, S. Mehta, J. T. Edsall, J. Biol. Chem. 1967, 242,
4221 – 4229. Compound 3 showed higher CA inhibitory activity
(IC50: 0.50 mm) than compound 4 (IC50: 1.6 mm). [Ru(bpy)3]Cl2
did not show a significant inhibition at 10 mm. See Figure S9 in
the Supporting Information.
[30] We examined the selective modification of CA in mouse
erythrocyte lysates at various concentrations of LSC 3 and
found that the selectivity of the modification at 1 mm was higher
than that at 10 mm, although the modification efficiency dropped.
Therefore, we carried out the further CA-selective modification
experiments in mouse erythrocyte lysates at 1 mm of 3.
[31] Q. Shao, B. Xing, Chem. Commun. 2012, 48, 1739 – 1741.
Keywords: photocatalysts · protein modifications ·
radical reactions · single-electron transfer · tyrosine
.
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